Characterization and Application of Bacteriophages and Endolysins as Biocontrol Agents to Combat Staphylococcus aureus

Abstract

Staphylococcus aureus is an opportunistic pathogen that causes several serious diseases in humans and animals ranging from skin infections to life-threatening diseases. In addition, it is considered as a major foodborne pathogen that can cause various symptoms of food poisoning. Especially, due to the emergence of multidrug-resistant S. aureus such as methicillin-resistant S. aureus (MRSA), S. aureus-targeting bacteriophages and endolysins have been proposed as alternative biocontrol agents to antibiotics. To develop a novel-type biocontrol agent against S. aureus, 70 phages were newly isolated and characterized. Mixtures of turbid and clear plaques were appeared after almost all the purified phages infection excluding two phages, suggesting that all the phages except SA12 and SA97 might be temperate phages. Phage SA12 was characterized and its genome was completely sequenced. Host range and bacteriophage challenge tests demonstrated its specific and efficient host lysis. As the phage SA12 formed lysogen when infected to the host bacterial strain, suggesting that the phage SA12 is a temperate phage. Meanwhile, phage SA97, which specifically inhibits the growth of S. aureus and produce only clear plaques, was characterized. Genome analysis revealed that the phage SA97 contains 40,592 bp of DNA encoding 54 predicted ORFs and none of these genes were related to virulence or drug resistance. Although a few genes associated with lysogen formation were detected in the phage SA97 genome, the phage SA97 produced neither lysogen nor transductant in S. aureus. When it was treated to milk, 4.15 log CFU/mL of S. aureus cells were reduced. These results suggest that the phage SA97 may be a promising candidate for controlling S. aureus. Despite the obvious advantages of virulent phages for biocontrol or therapy, the number of the virulent phages targeting S. aureus is limited. Therefore in this study, a virulent phage SA13m with mutated genes in the lysogen decision gene cluster was constructed, characterized and the genomes were compared with the wild-type phage SA13. First, S. aureus temperate phage SA13 was newly isolated, characterized, and the genome was sequenced and analyzed. The genomic DNA consists of 42,652-bp containing 62 ORFs with a lysogen gene cluster encoding integrase, phage repressor, CI, Cro, and antirepressor. To verify the lysogen formation by this gene cluster, a virulent phage SA13m with mutated genes in the cluster was developed and compared with SA13, suggesting that this gene cluster decides to form lysogen. Through the genome analysis of the phage SA13m, it was revealed that the genes in the lysogen decision gene cluster were considerably truncated and examined not to form lysogen. Moreover, the virulent phage SA13m rapidly killed the host cells compared to the temperate wild-type phage. In addition, the population of target bacteria was reduced to 4.33 log CFU/mL in 2 h incubation with the phage SA13m treatment to milk. From the results, the genetic engineering of a temperate phage to a virulent phage could be suggested and the virulent phage SA13m could be used as a promising biocontrol agent, as it has strong host lysis activity without lysogen formation. Bacteriophage endolysins, synthesized at the end of the phage life cycle, are the phage gene products that show antibacterial activities by hydrolyzing the peptidoglycan layer in bacterial cell wall. Comparison of 99 endolysin genes of S. aureus phages deposited in GenBank showed that they can be classified into six groups based on their domain composition. Interestingly, approximately 80.61% of the staphylococcal endolysins have a src-homology 3 (SH3_5) domain as a CBD, but the remaining 19.39%, including LysSA11 and LysSA97 endolysins, have a putative C-terminus CBD with no homology to the known one. In this study, two new kinds of endolysins LysSA11 and LysSA97 were purified and characterized. Bioinformatics analysis of LysSA11 endolysin, derived from a S. aureus virulent phage SA11, revealed an enzymatically active CHAP (cysteine, histidine-dependent amidohydrolases/peptidases) domain that showed an amidase activity. A novel CBD in C-terminus revealed to bind to the broad spectrum of staphylococcal cells. The activity was efficient in contaminated foods or utensils with MRSA. In particular, the populations of contaminated MRSA in milk and on ham were significantly reduced to 1.96-log CFU/mL and 3.37-log CFU/cm2, respectively, in 15 min. Moreover, polypropylene plastic cutting board and/or stainless steel knife with MRSA also showed a complete elimination about 4.01-log CFU/cm2 and/or 4.15-log CFU/cm2 reduction, respectively, in 30 min. This is the first report to assess the possibility of staphylococcal endolysin as an alternative biocontrol agent, not only as a food additive but also as a sterilizing agent. Further, the other endolysin LysSA97 was newly cloned from S. aureus phage SA97. Bioinformatics analysis revealed a novel putative CBD as well as two EADs containing CHAP and amidase_3 (N-acetylmuramoyl-L-alanine amidase) domains. The fusion protein containing green fluorescent protein and the putative CBD of LysSA97 endolysin showed a specific binding spectrum against staphylococcal cells suggesting that the C-terminal domain of LysSA97 endolysin is a novel CBD of staphylococcal endolysins. However, single treatment of LysSA97 endolysin showed weak bactericidal activities. In order to enhance its potential as a biocontrol agent against S. aureus, various kinds of essential oils were combined with LysSA97 endolysin. Among them tested, carvacrol with LysSA97 endolysin was found to have significant synergistic effect on the antimicrobial activity in food products including milk and beef. Conclusively, these results demonstrated that endolysin and carvacrol could act synergistically to inactivate Gram-positive bacteria such as S. aureus in food products. In this study, I suggested multiple approaches to control S. aureus by using bacteriophages and endolysins as biocontrol agents, and also investigated new CBDs for bioprobes.